﻿Two novel species and a new host record of Alternaria (Pleosporales, Pleosporaceae) from sunflower (Compositae) in Myanmar

﻿Abstract Sunflower (Helianthusannuus L.) is a widely cultivated, fast-growing crop known for its seeds and oil, with substantial ecological and economic importance globally. However, it faces challenges from leaf diseases caused by Alternaria species, which threaten its yield. Three small-spored Alternaria species were isolated from leaf spot and blight symptoms on sunflower in Myanmar. All the species were determined based on morphological characterization and a multi-locus phylogenetic assessment of seven genes, including the internal transcribed spacer of rDNA region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2), translation elongation factor 1-α (TEF1), Alternaria major allergen gene (Alt a 1), endopolygalacturonase gene (EndoPG), and an anonymous gene region (OPA10-2). The results introduced two new Alternaria species, A.myanmarensissp. nov. and A.yamethinensissp. nov., and a known species of A.burnsii, firstly reported from sunflower.


Introduction
The genus Alternaria Nees, 1816, which belongs to the family Pleosporaceae, order Pleosporales, and phylum Ascomycota, is a widely distributed dematiaceous fungus frequently found in plants, soil, food, and indoor air environments (Thomma 2003).It includes more than 790 species epithets, and approximately 382 species have been accepted (Hongsanan et al. 2020;Wijayawardene et al. 2020;Gannibal et al. 2022;Li et al. 2023;Liao et al. 2023).The identification and classification of Alternaria commonly rely on cultural features, conidial characteristics (shape, size, septation, beak formation), sporulation patterns, and hosts (Zhang 2003;Simmons 2007;Yu 2015).Normally, Alternaria is categorized into two obviously distinct groups: large-spored and small-spored Alternaria (Simmons 2007).The conidial bodies of large-spored species typically measure 60-100 μm in length and the small-spored species are less than 60 μm.The morphological criteria can be influenced by growth conditions, including substrate, light, and humidity, potentially undermining their reliability in characterizing the genus (Woudenberg et al. 2013).
Leaf spot and blight disease on sunflower (Helianthus annuus L.) caused by Alternaria significantly decreases head diameters and seed production (Kgatle et al. 2020).Sunflower, belonging to the Compositae family and native to North America, is an oilseed crop cultivated worldwide, with its oil ranking as the second most important source of edible vegetable oils (Zhang et al. 2021).The plant is also commercialized for livestock feed (Yegorov et al. 2019).It was introduced to Myanmar in 1968 (Favre and Myint 2009) and covered 0.224 million hectares with a yield of 9245 kg/ha in 2022 (http://faostat.fao.org/site/567/default.aspx-#ancor).In the Central Dry Zone of Myanmar (Mandalay, Sagaing, and Magway Regions), it contributes to more than 77% of the overall oilseed crop production (DOA 2020).During the monsoon season in 2023, three small-spored Alternaria species were isolated from leaf symptoms of sunflower collected in a plantation in Mandalay, Myanmar.In this study, those species were meticulously identified and illustrated through morphological and phylogenetic approaches.

Sample collection and fungal isolation
In August 2023, sunflower leaves displaying spot and blight symptoms were randomly collected from plantations in Myanmar, Mandalay Region, Yamethin Township, Segyi Village (30°21'28.188"N,112°08'32.136"E).From each field, samples were randomly collected at five different points, placed in separate clean zip bags and transported to the laboratory.For fungal isolation, leaf fragments from the edges of the lesions were excised, treated with a 1% sodium hypochlorite solution for three minutes, rinsed three times with distilled water, plated on moist filter papers in Petri dishes and then incubated at 25 °C in the dark for sporulation.A single spore was picked using a sterile glass needle under a stereomicroscope and inoculated onto potato dextrose agar (PDA: Difco, Montreal, Canada).Once sufficiently grown, pure cultures were isolated by a single spore and preserved in test tube slants at 4 °C in the Fungi Herbarium at Yangtze University (YZU) in Jingzhou, Hubei, China.MycoBank numbers were obtained by following the protocols outlined on (https://www.mycobank.org/).

Morphological characterization
To study the characteristics of the culture, mycelial plugs (6 mm diameter) were extracted from the periphery of 5-day-old colonies growing on PDA, transferred to fresh 90 mm PDA plates, and incubated in darkness at 25 °C for 7 days.For the examination of conidial morphology, mycelia were cultured on V8 juice agar (V8A) and potato carrot agar (PCA) under white fluorescent light at 22 °C with an 8-hour light/16-hour dark period (Simmons 2007).After a 7-day incubation period, the sporulation patterns and conidial characteristics were determined under an ECLIPSE Ni-U microscopic system (Nikon, Japan).The conidia were observed using a lactophenol-picric acid solution.Fifty randomly selected conidia were recorded.

DNA extraction, PCR amplification, and Sequencing
Genomic DNA extraction involved scraping fresh mycelia from colonies cultivated on PDA for 5 days at 25 °C, following the method outlined by Watanabe et al. (2010).Polymerase chain reaction (PCR) amplification and sequencing targeted specific genes of the internal transcribed spacer region of rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2), translation elongation factor 1-α (TEF1), Alternaria major allergen (Alt a 1), endopolygalacturonase gene (EndoPG), and an anonymous genomic region (OPA10-2).In the PCR processes, a 25 μL reaction mixture was prepared, consisting of 21 μL of 1.1× Taq PCR Star Mix (TSINGKE), 2 μL of template DNA, and 1 μL of each primer.The amplification reaction was performed using a Bio-Rad T100 thermocycler according to the conditions listed in (Table 1).The generated products underwent electrophoresis in a 1% agarose gel and were visualized by UV transillumination.Subsequently, the amplified products were purified and sequenced in both directions, facilitated by TSINGKE Company (Beijing, China).Initially, sequences from both ends were examined and manually edited using BioEdit v. 7.0.9(Hall 1999).Following this, the sequences were aligned and further edited with the PHYDIT v3.2 software (Chun 1995) before being submitted to GenBank (https://www.ncbi.nlm.nih.gov/)(Table 2).

Phylogenetic analysis
The resulting sequences were processed in the GenBank database at the National Center for Biotechnology Information (NCBI) using BLAST searches.The relevant sequences were downloaded and derived from newly reported sequences of recent publications (Woudenberg et al. 2015;Luo et al. 2018;Htun et al. 2022;Li et al. 2022Li et al. , 2023;;Romain et al. 2022) used in the present analysis (Table 2).The adjustments, alignments, and comparative analyses of the gene sequences were executed using ClustalX (Larkin et al. 2007) and Crandall 1998) with the Akaike Information Criterion (AIC).The "MrModelblock" file in MrModeltest was run using both the PAUP path (Swofford 2002) and the MrMt path (Nylander 2004).The two simultaneous Markov Chain Monte Carlo (MCMC) algorithms were launched from random trees, covering 10 6 generations, with data collected every 100 generations (Rannala and Yang 1996).The analysis was stopped when the standard deviation of split frequencies dropped below 0.01.A burn-in parameter of 25% was established, signifying that 75% of the trees were retained during the burn-in phase, with the remaining trees utilized for calculating the posterior probabilities in the majority-rule consensus tree.Subsequently, the phylogenetic tree was visualized and modified using Fig-Tree v. 1.4.3 (Rambaut 2016).In the phylogram, branch support is indicated by (posterior probability PP/bootstrap value BS) equal to or above 0.6/60%.

Phylogenetic analyses
The combined dataset, comprising sequences from seven gene loci (ITS, GAPDH, RPB2, TEF1, Alt a 1, EndoPG, and OPA10-2), included 59 Alternaria strains, containing the present 6 strains.It had 2,722 characters with gaps, allocated as follows: 466 characters for ITS, 302 for GAPDH, 307 for RPB2, 216 for TEF1, 421 for Alt a1, 391 for EndoPG, and 619 for OPA10-2.The phylogenetic tree was constructed and rooted using Alternaria alternantherae CBS 124392 as the outgroup.The Maximum Likelihood (ML) phylogeny was used as the foundational tree.Four strains fell into two independent clades and two, YZU 231747 and YZU 231748, were clustered with the strains of known species A. burnsii (Fig. 1).(Fig. 1).The results indicated that the current strains represented two new species and a known species of Alternaria, all belonging to the section Alternaria.
Notes.This species is phylogenetically grouped with A. koreana, A. orobanches, and A. ovoidea, based on sequences from ITS, GAPDH, RPB2, TEF1, Alt a 1, EndoPG, and OPA10-2 genes.It is distinct from A. koreana and A. ovoidea in its smaller conidial body size, particularly in width, and its sporulation patterns which produce catenulate conidia up to 6 units on PCA and V8A media, rather than those of the two closely related species (up to 2 units) on SNA and PDA (Table 3).Alternaria yamethinensis M.N.Zin & J.X. Deng, sp.nov.MycoBank No: 851391 Fig. 3 Etymology.The epithet designation is attributed to the Yamethin township, which was the location where the holotype was originally collected.
Notes. A. burnsii has been found in many countries on different hosts and substrates.The host range of A. burnsii is reported to include Apiaceae: Cuminum cyminum (Uppal et al. 1938), Bunium persicum (Mondal et al. 2002), Apium graveolens (Zhang 2003;Zhuang 2005), Cumin (Shekhawat et al. 2013 (Simmons 2007;Wei et al. 2022).In this study, three Alternaria species associated with sunflower in Myanmar have been identified, and pathogenicity tests reveal that these present Alternaria species are causal pathogens for sunflower, of which A. yamethinensis sp.nov. is identified as the most pathogenic one (Suppl.material 1).This discovery underscores the importance of Alternaria leaf spot and blight on sunflower and helps in disease management in Myanmar.

Figure 1 .
Figure 1.Phylogenetic tree generated from maximum likelihood analyses using aligned ITS, GAPDH, RPB2, TEF1, Alt a 1, EndoPG, and OPA10-2 gene sequences of the present Alternaria strains and their related species.Bootstrap support (BS) values ≥ 60% and Bayesian posterior probability (PP) scores ≥ 0.60 were shown at the nodes (ML/PP).Alternaria alternantherae CBS 124392 was used as an outgroup.Type strains are marked 'T'.Representative strains are marked 'R'.The strains from the present study are highlighted in bold.

Table 1 .
Primers and PCR protocols.

Table 2 .
The GenBank accession numbers of Alternaria strains used in the present study.

Table 3 .
Morphological comparison of the present Alternaria and their relevant species.